Characterization of RAFTK, a novel focal adhesion kinase, and its integrin-dependent phosphorylation and activation in megakaryocytes

We have recently isolated a cDNA encoding a novel human intracellular tyrosine kinase, termed RAFTK (for a related adhesion focal tyrosine kinase). The RAFTK cDNA, which encodes a polypeptide of 1,009 amino acids, shares 65% homology to the focal adhesion kinase (FAK), including several consensus motifs. In this report, we describe the biochemical characterization and functional analysis of the RAFTK protein. Coexpression of RAFTK and FAK proteins in megakaryocytic cells and blood platelets was observed. Using a specific antibody to RAFTK and the monoclonal antibody 2A7 to FAK, FAK and RAFTK could be distinguished antigenically. RAFTK had intrinsic tyrosine kinase and autokinase activities. It was phosphorylated on tyrosine in growing cultures of COS cells transfected with the pCDNAIII/flag-RAFTK expression vector containing the RAFTK cDNA ligated with the 8 amino acid flag peptide sequence. Similar to FAK, dephosphorylation of RAFTK was observed when adherent transfected COS cells were detached. Phosphorylation was regained upon replating of these cells on the fibronectincoated dishes. Analysis of tyrosine-phosphorylated RAFTK from adherent transfected COS cells showed that the Src homology 2 (SH2) domains of the Src and Fyn protein kinases as well as the Grb2 adaptor protein were able to specifically associate with RAFTK. Tyrosine phosphorylation of endogenous RAFTK was observed upon fibronectin-induced activation of human megakaryocytic cells. Furthermore, colocalization of RAFTK protein with vinculin, a focal adhesion protein, was observed by confocal microscopy in focal adhesion- like structures in adherent CMK cells and in transfected pCDNAIII/flag- RAFTK COS cells upon fibronectin activation. These data suggest that RAFTK is a novel member of the FAK family, that it localizes to focal adhesion-like structures in CMK megakaryocytic cells, that it participates in integrinmediated signaling pathways in megakaryocytes, and that it is able to associate with the tyrosine kinases Src and Fyn as well as the adaptor protein Grb2 via SH2-phosphotyrosine interactions.     

Randomised trial comparing the upright and supine positions for the second stage of labour

Objective To assess the maternal and neonatal effects of upright compared with recumbent positions during delivery, in terms of defined outcome variables.
Design A randomised controlled trial.
Setting St Monica's Nursing Home, a midwife based maternity unit in Cape Town, South Africa.
Participants Five hundred and seventeen women of low obstetrical risk assigned to deliver at the nursing home.
Results The trial showed that women who adopted the upright posture for delivery experienced less pain, perineal trauma and fewer episiotomies than those who delivered in the supine position.
Conclusion The data suggest that in women of low obstetrical risk, choice of posture during delivery may be encouraged.     

Giant collagen plaques in Creutzfeld-Jakob disease

Summary We report two cases of Creutzfeld-Jakob disease with clusters of giant collagen fibers. To our knowledge, these abnormally large collagen fibers have never been described in patients with degenerative diseases of the central nervous system. The significance of the formation of such plaque-like large collagen fibers has as yet not been elucidated. It is felt that these represent a product of the degenerative process.Presented in part at the 68th Annual Meeting of the American Association of Neuropathologists, St. Louis, Missouri, June 18–21, 1992     

Introns and gene evolution

In one scenario of gene evolution, exon shuffling has a fundamental role in increasing gene diversity. As DNA sequences accumulate in the databases, the picture of the intron/exon structures of genes becomes more and more clear. We discuss in this review some features of this picture that suggest that introns have been present since the early stages of evolution, and that exon shuffling was a fundamental process in the construction of ancient as well as modern genes.     

Validity of a Brazilian version of the Older Americans Resources and Services (OARS) mental health screening questionnaire

A validity study of the Brazilian version of the 15-item Short Psychiatric Evaluation Schedule (SPES), included in the mental health assessment of Older Americans Resources and Services (OARS), designed to detect psychiatric disorders in the elderly, against the "caseness" criterion suggested by Cooper and Schwarz was carried out with a community sample, as part of a survey to study health and living conditions of the elderly in a large urban center of a developing country, S?o Paulo, Brazil. The screening questionnaire was completed by 292 subjects, and 91 were selected for the psychiatric interview. The validity coefficients were as follows: sensitivity 61%, specificity 89%, positive predictive value 66%, negative predictive value 87% and misclassification rate 18%. A discriminant analysis using a stepwise procedure was then applied to select the best item discriminators of the screening questionnaire. The best set of discrimination comprised six items leading to the following validity coefficients: sensitivity 82%, specificity 77%, positive predictive value 58%, negative predictive value 92% and misclassification rate 21%. The possible factors related to false positive and false negative responses on the screening are discussed.     

Laparoscopic findings in serosal eosinophilic gastroenteritis. Report of two cases

This report describes the laparoscopic features of two patients with serosal eosinophilic gastroenteritis. In one case only hyperemia of the peritoneal serosa was found. In the other the laparoscopic picture resembled peritoneal carcinomatosis. We show that laparoscopy and peritoneal biopsy may permit diagnosis of the disease without the need for laparotomy.     

Echo-guided percutaneous puncture: a safe and valuable therapeutic tool for amebic liver abscess

To clarify the therapeutic role of echo-guided percutaneous puncture (EPP) in management of amebic liver abscess, 20 patients (24 abscesses) received metronidazole plus EPP. Fluid was aspirated through Chiba needles under real-time sonographic guidance so as to reduce cavity size to less than 3 cm. Not more than two EPPs were necessary in the majority of cases and no complication followed the procedure. This scheme resulted in a shortening of time of both hospitalization (less than or equal to 20 days) and liver lesion healing as assessed by ultrasound (less than or equal to 4 months). It is concluded that EPP is a valuable and safe therapeutic tool for hepatic amebic abscess.     

Recombinant human interferon-alpha induced cytoreduction in chronic myelogenous leukemia

Summary Fourteen patients with Ph'-chromosome positive chronic myelogenous leukemia (CML) in first chronic phase were treated with recombinant interferon-_2c. Interferon-_2c 5 to 10×10⁶ units s.c. was given for 12 weeks as an induction therapy. Maintenance treatment consisted of interferon-_2c 5 × 10⁶ units twice weekly s.c.. Two patients (14%) attained a complete clinical remission and 6 (43%) a partial remission, 3 of whom developed progressive disease during maintenance therapy. A complete disappearance of Ph'-chromosome was achieved in 1 patient. All patients had a more than 45% initial decline of the leukocyte count. Four out of ten patients with an initially enlarged spleen demonstrated reduction in spleen size. Influenza-like symptoms, anorexia, nausea, weight loss and fatigue were common side effects. Interferon-alpha is active in CML but additional clinical investigations are warranted to assess more precisely the therapeutic value of the interferons in this disease.     

High-frequency mutations in a plasmid-encoded gas vesicle gene in Halobacterium halobium

Gas vesicle-deficient mutants of Halobacterium halobium arise spontaneously at high frequency (about 1%). The mutants are readily detected, forming translucent colonies on agar plates in contrast to opaque wild-type colonies. To investigate the mechanism of this mutation, we recently cloned a plasmid-encoded gas vesicle protein gene, gvpA, from H. halobium. In the wild-type NRC-1 strain the gvpA gene is encoded by a multicopy plasmid of approximately 150 kilobase pairs (kb). We have now characterized 18 gas vesicle-deficient mutants and 4 revertants by phenotypic and Southern hybridization analyses. Our results indicate that the mutants fall into three major classes. Class I mutants are partially gas vesicle-deficient (Vac(delta-)) and unstable, giving rise to completely gas vesicle-deficient (Vac(-)) derivatives and Vac(+) revertants at frequencies of 1-5%. The restriction map of the gvpA gene region in class I mutants is unchanged but the gene copy number is reduced compared to the Vac(+) strains. Class II mutants can be either Vac(delta-) or completely Vac(-) but are relatively stable. They contain insertion sequences within or upstream of the gvpA gene. A Vac(-) class II mutant, R1, contains the 1.3-kb insertion sequence, ISH3, within the gvpA gene, whereas four Vac(delta-) class II mutants contain other insertion sequences upstream of the gene. Class III mutants are stable Vac(-) derivatives of either the wild-type or class I mutants and have no detectable copies of the gvpA gene. Based on these results, we discuss the mechanisms of gas vesicle mutations in H. halobium.